1. Upon depolarization, voltage-gated sodium channels assume non-conducting inactivated states which may be characterized as "fast' or "slow' depending on the length of the repolarization period needed for recovery. Skeletal muscle Na+ channel alpha-subunits expressed in Xenopus laevis oocytes display anomalous gating behaviour, with substantial slow inactivation after brief depolarizations. We exploited this kinetic behaviour to examine the structural basis for slow inactivation. 2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusion of the pore by III-IV linker residues, the structural features of slow inactivation are unknown. Since external pore-lining residues modulate C-type inactivation in potassium channels, we performed serial cysteine mutagenesis in the permeation loop (P-loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residues are involved in Na+ channel slow inactivation. 3. Wild-type and mutant alpha-subunits were heterologously expressed in Xenopus oocytes, and Na+ currents were recorded using a two-electrode voltage clamp. Slow inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptophan residues in domains II, III and IV did not alter slow inactivation. 4. Analogous to the W402C mutation, coexpression of the wild-type alpha-subunit with rat brain Na+ channel beta 1-subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta 1-subunit coexpression did not. We propose that the W402C mutation and the beta 1-subunit modulate gating through distinct mechanisms. 5. Removal of fast inactivation in wild-type alpha-subunits with the III-IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed the development of slow inactivation. We propose that slow inactivation in Na+ channels involves conformational changes in the external pore. Mutations that affect fast and slow inactivation appear to interact despite their remote positions in the channel.